U.S. patent application number 14/383332 was filed with the patent office on 2015-02-19 for formalin-free fixation agent for histological stains of tissue samples.
The applicant listed for this patent is Roberto Gerigk. Invention is credited to Roberto Gerigk, Michael Gudo.
Application Number | 20150050689 14/383332 |
Document ID | / |
Family ID | 47998392 |
Filed Date | 2015-02-19 |
United States Patent
Application |
20150050689 |
Kind Code |
A1 |
Gerigk; Roberto ; et
al. |
February 19, 2015 |
Formalin-Free Fixation Agent For Histological Stains of Tissue
Samples
Abstract
A fixation agent including at least one polyamine and at least
one acidification agent. The quality and staining of tissue samples
or tissue sections that have not been fixed with formalin is
improved, with higher brilliance and color stability of the stain,
allowing the tissue to be cut with a better quality without
artifacts.
Inventors: |
Gerigk; Roberto; (Muhldorf
am Inn, DE) ; Gudo; Michael; (Maintal, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Gerigk; Roberto |
Munich |
|
DE |
|
|
Family ID: |
47998392 |
Appl. No.: |
14/383332 |
Filed: |
March 1, 2013 |
PCT Filed: |
March 1, 2013 |
PCT NO: |
PCT/EP2013/054146 |
371 Date: |
September 5, 2014 |
Current U.S.
Class: |
435/40.52 |
Current CPC
Class: |
G01N 1/36 20130101; G01N
1/30 20130101 |
Class at
Publication: |
435/40.52 |
International
Class: |
G01N 1/36 20060101
G01N001/36 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 6, 2012 |
DE |
102012101896.0 |
Claims
1. A fixation agent for tissue samples, comprising: at least one
polyamine, selected from the group consisting of urotropine,
dimethylol dihydroxyethylene urea, mono-, di-, tri-, tetra-, penta-
or hexamethylol melamine, tetramethylol acetylene diurea,
dimethylol propylene urea, acetoguanamine or 5,5-dimethylhydantoin,
and mixtures thereof, and at least one acidification agent selected
from the group of free monovalent, bivalent or polyvalent acid
consisting of oxalic acid, fumaric acid, tartaric acid, maleic
acid, succinic acid, hydrochloric acid, acetic acid, propionic
acid, formic acid, mono-, di- or tri chloroacetic acid or
chloroacetic acid, boric acid, phosphoric acid, and mixtures
thereof, wherein the polyamine reacts with protons released from
the acidification agent, thus forming or releasing aldehydes, and
the molar ratio between the molar concentration of releasable
aldehyde per mol of amine to the molar concentration of releasable
protons per mol of acid lies in a range from 1:0.7 to 1:1.5.
2. The fixation agent according to claim 1, further including at
least one further polyfunctional aldehyde selected from the group
consisting of citral, 3,7-dimethylocta-2,6-dienal, geranial,
(E)-3,7-dimethylocta-2,6-dienal, propanal, butanal, valeric
aldehyde, pentanal, hexanal, heptanal, octanal, nonanal, decanal,
(2 E)-3-phenylprop-2-enal, benzaldehyde, phenylmethanal, vanillin
aldehyde, 4-hydroxy-3-methoxyphenylmethanal, and mixtures
thereof.
3. The fixation agent according to claim 1, wherein the molar
concentration of the total available aldehyde in the solution is at
least 0.5 mol/l.
4. The fixation agent according to claim 1, wherein it is present
in aqueous solution.
5. The fixation agent according to claim 1, wherein it is set to a
pH range of 3-8.
6. The fixation agent according to claim 1, further including at
least one strongly hydrophilic compound selected from the group
consisting of monopropylene glycol, dipropylene glycol,
polypropylene glycol, glycerol, pentaerythritol, sorbitol, ethylene
glycol, diethylene glycol and polyethylene glycol.
7. The fixation agent according to claim 1, further including at
least one surfactant selected from the group consisting of
ethoxylated non-ionic surfactants with high and/or low HLB value,
polysorbates, particularly polysorbate 20, 40, 60 or 80, saponins,
alkali salts of decyl sulfates, decyl sulfonates, dodecyl sulfates,
dodecyl sulfonates and dodecylbezenesulfonates, oleates, stearates,
caprates, caprylates and betaines.
8. The fixation agent according to claim 1, wherein it is present
as anhydrous mixture of crystalline or anhydrous components, which
are added in the form of soluble powder or as soluble pressed
tablets to a suitable solvent.
9. A method for staining tissue samples fixed in a formalin-free
manner, comprising: in a first step the tissue sample is fixed with
the fixation agent according to claim 1, in a second step the
tissue sample is prepared for staining, in a third step the tissue
sample is stained, and is evaluated microscopically.
10. Use of the fixation agent according to claim 1, for preparing
and fixing tissues samples for histological or immunohistochemical
stains of the tissue sample.
11. The method of claim 9 wherein the second step includes cutting
the tissue sample.
12. The use of the fixation agent according to claim 10 without
utilizing formalin.
Description
[0001] The invention relates to a development of a fixation
solution which effects a clear improvement of the known
histological and immunological staining techniques as well as the
staining techniques used in pathology. In particular, the present
invention improves the quality and staining of tissue samples or
tissue sections that have not been fixed with formalin. The present
invention has proven to be especially advantageous for sensitive
tissue that is difficult to cut when fixed with formalin, which can
not only be cut with a better quality without artefacts, but also
has a higher brilliance and colour stability of the stain.
[0002] For histological examinations in biology and medicine,
tissue samples are removed, usually fresh, from an organism, or
even entire organs or entire animals/animal bodies/plants or
organisms, are placed in special solutions known as fixation
solutions (infiltration) or are saturated with the fixation
solution via a perfusion or instillation arrangement. The fixation
serves to fix (=preserve) the state of the tissue at the moment of
removal in order to examine this state directly microscopically at
a later moment in time and/or to stain it and then microscopically
differentiate the stained cell or tissue parts on the basis of
their chemical properties and therefore examine these cell or
tissue parts or to provide diagnoses.
[0003] If a tissue sample is placed in the fixation solution,
reference is made to an "immersion fixation", and if the tissue
piece or organ or the entire organism is saturated via its vascular
system with the fixation solution, reference is made to perfusion
or instillation fixation. The fixation process is a chemical
reaction of the actual fixation agent with the proteins of the
tissue. A sample is thus preserved, that is to say decomposition
processes by enzymes, bacteria or fungi are prevented. In addition,
the tissue is hardened and is now converted into a state in which
it can be cut using a microtome into thin slices a few .mu.m
thick.
[0004] Subsequently to the fixation, the tissue sample can be
embedded in paraffin, for example. To this end, the sample is
usually firstly dewatered in an alcohol series with increasing
concentrations. The alcohol is then removed via what is known as an
intermedium, the sample is then saturated with paraffin via xylene
or a xylene replacement in a number of steps, and lastly is cast in
paraffin by means of a mould. Once the paraffin has solidified, the
paraffin block thus obtained is processed using a microtome in
order to provide thin sections (generally between 2 and 5 .mu.m
thick).
[0005] A paraffin block can be stored in a practically unlimited
manner under room temperature conditions or slightly cooled, that
is to say for example individual sections can be produced, and the
block can then be stored again and kept for subsequent further
processing.
[0006] Instead of paraffin, other polymers can also be used for the
embedding of samples, for example celloidin, high-molecular PEG,
acrylates and others.
[0007] Such thin sections are usually arranged on glass slides and
then stained in accordance with specific instructions (what are
known as histological staining protocols or staining provisions).
Only as a result of the staining is it possible to microscopically
differentiate differences in the tissue structure, distinguish
diseased tissue from healthy tissue, and perform diagnostic
assessments, since the stains used bind specifically to certain
structures or chemical components of the tissue structures and
therefore stain these specifically, that is to say depending on
their chemism. For example, cell nuclei can thus be differentiated
significantly from cell plasma, and various cell types are stained
differently as well as fibres and other tissue components. The
overall image of a stain then allows a diagnostic assessment of the
examined tissue.
[0008] The most conventional fixation agent in histology is
formalin. It was used for the first time as fixation agent for
tissue samples by Isaak and Ferdinand Blum at the Senckenberg
research institute in Frankfurt am Main in the early 1890s and,
from the outset, proved to be much better suited, for example
compared with spiritus (ethanol), turpentine and other substances
(see I. Blum: 1893). Formalin very quickly became widespread in the
field of medicine and ultimately established itself globally as the
ultimate fixation agent, because it was easy to acquire and easy to
handle, and the results are comparable with one another over a
broad field of application.
[0009] Besides formalin, many further fixation agents were, and
are, also used in histology that are based on methanol, ethanol,
acetic acid, chloroform, chloral hydrate, picric acid, mercury
dichloride, potassium dichromate, chromic acid, osmium tetroxide
and various salt solutions and salt mixtures. However, these
fixation agents are not all suitable universally, but in each case
only specifically for certain tissue types and certain questions
and further processing. There is a direct link between fixation
(fixation agent), tissue preservation and result of the stain,
which ultimately can be evaluated by microscope. Since the fixation
in the majority of cases constitutes a chemical reaction (in this
respect see "Histological Techniques, Laboratory Columbia Manual
University", 1975), which influences the chemical properties of the
tissue, it has long been known already that certain dyes or certain
dye solutions are only compatible with certain fixation agents or
fixation methods or are incompatible therewith. When, for example,
fixation is performed using ethanol or using ethanol/acetic acid
mixtures, tissue differentiations can in principle only still be
performed with a great deal of difficulty, both because cells and
tissue are severely dehydrated in the case of ethanol fixation and
shrink, and also because mordant dyes for example do not provide
satisfactory results. On the other hand, stains with use for
example of picric acid have a much more brilliant and vibrant
manifestation than with use of formalin as fixation agent, and
therefore pre-treatments of the sections with picric acid are even
recommended in some staining protocols.
[0010] Although formalin was initially distinguished as an
excellent fixation agent, it was observed over time that some
problems occurred with numerous staining techniques in histology
and pathology. Since formaldehyde oxidises in solution and in
contact with the air to form formic acid, what are known as
"formalin pigments" form in blood-rich samples. These highly
refringent crystals are formed from the reaction of formic acid and
blood and are perceived in histology and pathology as interfering
artefacts. Stabilisers against the polymerisation, which are added
to the commercial formalin solution, such as methanol and butanol,
may also negatively influence the results of histological staining,
since they act in a dehydrating manner (similarly to ethanol) and
interfere with some dye/tissue bonds.
[0011] Formalin also plays a central role in a special branch of
histology, which is known as immunohistochemistry. Here, however,
formalin is not generally used as "formalin 4%", but as
"paraformaldehyde solution". Paraformaldehyde (PFA) is the
polymerised form of formalin, which is obtainable in the industry
as powder and can be dissolved completely in hot, alkaline
solution. Formalin, (the aqueous solution of formaldehyde gas) has
the property in aqueous solution of forming chains of
paraformaldehyde, which can precipitate. Furthermore, formaldehyde
decomposes under the action of light, heat and/or oxygen to form
formic acid. In an aqueous formalin solution, degradation and
polymerisation reactions take place constantly, as a result of
which the formaldehyde is broken down continuously. Since there are
also reformation reactions (depolymerisation of the PFA chains, and
also balanced reactions), the percentage of formaldehyde in an
aqueous solution can never be specified exactly.
[0012] The "stock solution", which is known under the trade name
"Formol", has a formaldehyde concentration of 35-37%, sometimes
also 37-38%. Formol is offered in various qualities: technical,
stabilised and buffered. Here, additives are admixed to this
saturated formaldehyde solution: stabilised Formol is stabilised
with methanol (in order to prevent PFA formation), and buffered
Formol is buffered with calcium carbonate, borax or a phosphate
mixture (against the reduction of the pH value in the event of
decomposition to form formic acid). A 4% formalin solution is then
produced from this stock solution by means of dilution
(approximately 1+9) and is consequently not exactly 4%, but rather
3.5-3.9%. Some producers offer 4% formalin as "actual" 4% formalin,
that is to say the stock solution is not diluted 1+9, but is
diluted in accordance with actual percentages by mass.
[0013] Nevertheless, the result is also a solution in which the
content of formalin fluctuates and reduces successively. For
"normal" histological questions, this is largely unproblematic, but
for immunohistochemical questions this fluctuating composition,
which may additionally have a fluctuating pH value due to the
formation of formic acid, may be disadvantageous. Some histological
stains are also negatively influenced by an excessively low pH
value of formalin (for example the differentiation of oxidative and
glycolytic muscle fibres). A fixation solution with exact
concentrations of 4%, 6% or 8%, which is adjusted via a buffer to
an exact pH value (for example 7.0, 7.2 or 7.4, which corresponds
to a conventional pH value of animal/human tissue), is therefore
produced from paraformaldehyde for IHC questions. However, such
buffered PFA solutions only last for a short period of time, that
is to say they have to be used up within a few days, because
otherwise exactly the same decomposition and balanced reactions as
with normal formalin solution take place. For IHC questions, in
which the degree of cross-linking of the proteins with one another
plays a key role, buffered PFA is therefore used, because in this
case more uniform and more reproducible fixation and reaction
conditions are attained.
[0014] The disadvantages and inadequacies of formalin (and also of
PFA) have long been known and lie not only in their pungent, sharp
smell, which is bothersome and unpleasant, but also constitute a
significant health risk according to more recent findings. In the
EU, a list of the dangerous chemicals (VHCCs or very high concern
chemicals) has been compiled and updated within the scope of the
REACH Regulation (EC Regulation 1907/2006, with amendments in EC
No. 1354/2007 and 1272/2008, implemented in German law in the
Chemicals Act of 2 Jul. 2008), and measures have been adopted as to
when and how such VHCCs are to be avoided in future or use thereof
limited or replaced.
[0015] A further disadvantage of formalin-fixed tissue pieces is
the fact that such tissue pieces tend to harden over long periods
of storage, which has a negative effect on the cuttability and
stainability of the tissue. Generally, some tissues that have been
fixed in formalin become brittle and cracked and can hardly be cut
or can only be cut with difficulty (for example liver, kidneys,
spleen, muscles, nerve tissue, brain).
[0016] A further negative of formalin is the shrinkage and the
dissolving out of fats and glycogen. A shrinkage of up to 10% of
the tissue volume is normal in the case of formalin fixations. This
leads to cracks in the tissue and also to some sort of
misinterpretation in terms of the expansion and size of certain
structures.
[0017] Considered on the whole, formalin and also buffered PFA in
an overall view of the known fixation agents are not the fixation
agents that deliver the best results, but rather the fixation
agents that deliver comparable results due to the many years of
experience and that could be used and acquired
cost-effectively.
[0018] Another variant of examining tissue in a state that is as
fresh as possible is the frozen section. Here, the state is fixed
by immediate freezing following the sample removal. Here, however,
there is no cross-linking of the proteins with one another. The
sample will only last as long as it is held at corresponding low
temperature. In addition, the freezing has to be implemented
suddenly to very low temperatures (generally <-78.degree. C.),
because otherwise the formation of water crystals could destroy
fine structures of the tissue. The "frozen section" is what is
known as the quick section and is generally produced only for
special applications in which, for example, there is no time to
wait for the chemical fixation reaction (for example quick
diagnostics during an operation) or when the stain or an
immunohistochemical detection has been disturbed from the outset by
the fixation itself. Under these conditions, a diagnostic decision
can be made with the disadvantages of the frozen section.
[0019] However, experience generally indicates that stains on fresh
tissue are often more intensive and vibrant and that staining times
have to be reduced. Where possible, this is due to the fact that
the binding of the dyes often is not coupled to the chemical
changes caused by the fixation, but because the dyes bind to the
tissue components themselves. The chemical fixation reaction can
thus be identified here in wide areas as being disruptive for a
stain that provides good differentiation.
[0020] Consequently, the frozen section would be the qualitatively
better approach for many stains if it were not so difficult to
handle and to store.
[0021] The disadvantages in the case of frozen sections include,
for example, the technical outlay of the conservation (immediate
freezing following removal), the problem of longer-term storage
(the storage temperature may not be exceeded), the relatively thick
sections (generally around 10 .mu.m), the impossibility of
decalcifying a frozen section, etc. For this reason, the fixation
of a tissue sample is a necessary step in order to ensure permanent
and longer-term conservation.
[0022] For these reasons, there is a considerable need to provide
further alternatives that allow comparable or even better
stainability of tissue samples and sections, and therefore improve
the diagnostic research.
[0023] The provision according to the invention of an alternative
fixation agent for tissue samples of all types, in particular for
histology and immunohistochemistry, is based precisely on this
overall situation and confronts the problem of ensuring that tissue
samples of all types, with use of the fixation according to the
invention, and also all staining techniques are accessible and
enable at least comparable, if not better staining success.
[0024] To this end, the invention provides a formalin-free
solution, which is suitable for fixing tissue samples of all types
and for improving the stainability of the samples. The solution
according to the invention is characterised by the fact that, due
to the acid present in the composition according to the invention,
a total aldehyde concentration of at least 0.5 mol/l, but where
necessary also considerably more, can be released.
[0025] To this end, the solution according to the invention
includes the components of claim 1. Preferred embodiments or
applications are formulated in the dependent claims.
[0026] In particular, the solution according to the invention
contains [0027] at least one polyamine, selected from the group
consisting of urotropine triazines, mono-, di-, tri-, tetra-,
penta- or hexamethylol melamine, dimethylol-dihydroxyethylene urea,
tetramethylol acetylene diurea, dimethylol propylene urea,
acetoguanamine or 5,5-dimethylhydantoin, and mixtures thereof, and
[0028] at least one acidifier selected from the group of free
bivalent or polyvalent acids consisting of oxalic acid, fumaric
acid, tartaric acid, maleic acid, succinic acid, hydrochloric acid,
acetic acid, propionic acid, formic acid, mono-, di- or
trichloroacetic acid or chloroacetic acid, boric acid, phosphoric
acid, or mixtures thereof, characterised in that the polyamine
reacts with the protons released by the acid, thus forming
aldehydes. Compositions that for example form formaldehyde,
glyoxal, glutaraldehyde, ethanal or propanal and where applicable
are able to release these are particularly preferred.
[0029] The molar ratio between concentration of the maximum
releasable aldehydes in mol to the concentration of the maximum
releasable protons in mol is 1:0.7 to 1:1.5, preferably 1:0.7 to
1:0.9, more preferably 1:0.8 to 1:1, more preferably 1:0.9 to
1:1.2, more preferably 1:1 to 1:1.4 and/or more preferably 1:1.2 to
1:1.5 in the solution according to the invention and is therefore
selected such that a ratio of at least 0.5 and at most 2 is set
between "total releasable aldehyde" and "total protons releasable
from the acid".
[0030] The solution according to the invention is further
characterised in that the solution comprises at least one further
polyfunctional aldehyde selected from the group consisting of
citral, 3,7-dimethylocta-2,6-dienal, geranial,
(E)-3,7-dimethlyocta-2,6-dienal, propanal, butanal, valeric
aldehyde, pentanal, hexanal, heptanal, octanal, nonanal, decanal (2
E)-3-phenylprop-2-enal, benzaldehyde, phenylmethanal, vanillin
aldehyde, 4-hydroxy-3-methoxyphenylmethanal, and mixtures
thereof.
[0031] These compounds are preferably aldehydes that still have one
or more additional chemical functional groups. These chemical
functional groups can be selected from the following: aryl groups,
whether phenyl, naphthyl, thienyl, indolyl, etc., linear or
branched alkyl, alkenyl or alkyne groups, and also corresponding
halogenated groups; groups with oxygen hydroxyl, carbonyl,
aldehyde, halogen formyl, carbonate ester, carboxylate, carboxyl,
ester, hydroxyperoxy, hydroxy, ether; groups with nitrogen, such as
carboxamide, amines, imines, imides, azides, azo, cyanates,
nitrates, nitrile, nitrosooxy, nitroso and pyridyl; groups with
sulfur such as sulfhydryl, sulfides, disulfides, sulfinyl,
sulfonyl, sulfino, sulfates, thiocyanates, carbonothioyl; groups
with phosphorous, such as phosphino, phosphono, phosphate.
[0032] These groups can occur individually or in multiple, or may
also be present in combinations with one another.
[0033] Here, the aldehydes for example are present with one or more
functions selected from the above-mentioned groups, or what are
known as multi-functional aldehydes, because they can bind by means
of their aldehyde function to various points of the sample via
different chemical mechanisms and provide via their other chemical
functions, incorporated and included inherently, docking points for
the utilised dyes, which are used in order to stain the sample. Due
to the addition of polyfunctional aldehydes, additional chemical
functional groups will therefore bind to the tissue sample during
the step of tissue fixation.
[0034] The chemical functions of the multi-functional aldehydes are
selected such that certain types of dyes are bound better than
others or are also selected in such a way that certain types of
dyes are prevented or discouraged from binding to the sample. Due
to the different affinity of the sample, which is also referred to
as differentiated chemism of the different tissues, a spatial
differentiation of the stainability of the sample is thus attained.
The control mechanism thus attained is an advantage of the
invention, by means of which the colour contrast of the sample by
the addition of multi-functional aldehydes is additionally
controlled, because additional docking points and reaction points
are provided for the molecules of the dyes due to the binding of
these functional groups to the tissue sample, such that an optimal
staining can not only be achieved more quickly, but in particular a
higher colour intensity, brilliance and/or contrast can also be
attained.
[0035] In accordance with a further embodiment, a further fixation
solution in the context of this invention contains hexamethylol
melamine, boric acid, sodium hydroxide, phenylmethanal, Tween 20,
wherein hexamethylol melamine is a formaldehyde cleaver, and
phenylmethanal serves as polyfunctional aldehyde. The practical
implementation for producing this composition will be discussed in
the examples.
[0036] The molar concentration of the total available aldehyde in
the solution according to the invention, which is formed from the
polyamine together with the acid, or which is produced by the
polyfunctional aldehyde, is preferably set to at least 0.5 mol/l,
preferably 0.6-0.69 mol/l, more preferably 0.7-0.79 mol/l, more
preferably 0.8-0.89 mol/l, more preferably 0.9-0.99 mol/l, more
preferably 0.99-1.2 mol/l, in the solution according to the
invention. With such a solution, an optimal fixation of tissue
samples is ensured, which improves both the cuttability of the
sample and also has a positive influence on the stainability of the
sample.
[0037] In order to prevent the sample from drying out, a highly
hygroscopic chemical can be added to the sample as further
additive, which is selected from the group containing monopropylene
glycol, dipropylene glycol, polypropylene glycol, glycerol,
pentaerythritol, sorbitol, ethylene glycol, diethylene glycol and
polyethylene glycol.
[0038] In order to reduce the surface tension of the solution and
to improve the creep properties, a surfactant can be added to the
solution as further additive, which is selected from the group
containing ethoxylated non-ionic surfactants with high and/or low
HLB value, polysorbates, particularly polysorbate 20, 40, 60 or 80,
saponins, alkali salts of decyl sulfates, decyl sulfonates, dodecyl
sulfates, dodecyl sulfonates, dodecylbezenesulfonates, oleates,
stearates, caprates, caprylates and betaines.
[0039] In order to adapt the isotony and osmolarity of the
solution, organic or inorganic salts can also be used, which are
selected from the group of lithium chlorides, sodium chlorides,
potassium chlorides, calcium chlorides, strontium chlorides,
lithium sulfates, sodium sulfates, potassium sulfates, calcium
sulfates, strontium sulfates, lithium acetates, sodium acetates,
potassium acetates, calcium acetates, strontium acetates, lithium
citrates, sodium citrates, potassium citrates, calcium citrates,
strontium citrates, lithium nitrates, sodium nitrates, potassium
nitrates, calcium nitrates, strontium nitrates, lithium succinates,
sodium succinates, potassium succinates, calcium succinates,
strontium succinates and/or lithium formates, sodium formates,
potassium formates, calcium formates, strontium formates.
[0040] In order to adapt the flow properties of the solution,
organic or inorganic thickening agents can also be used, which are
selected from the group of carbomers, starch and modified starch,
agarose, dextrose, methyl cellulose, ethyl cellulose or propyl
cellulose, acrylic acid and PVA.
[0041] In accordance with a further embodiment, the declared
solution is present in aqueous form. Furthermore, in accordance
with a further embodiment, the components of the declared solution
are provided as an anhydrous mixture of crystalline and/or
anhydrous components, which are provided in the form of soluble
powder or as soluble, pressed tablets. This powder or these tablets
is/are dissolved by the addition of water or another suitable
solvent or mixture thereof in order to provide a fixation solution
that is ready for use.
[0042] An advantage of the solution according to the invention is
also the pH stability thereof. The solution according to the
invention is stable in pH ranges of pH 3-8, preferably pH 3-6. This
stability is achieved by the adjustment of the molar ratio of the
amine group to the acid groups in a ratio of approximately 1:1.
Provided free polyamine is present in the solution, this functions
as deposit, and the pH cannot rise. This pH stability is based on
the strong buffer capacity of the polyamines, which generally react
in a slightly alkaline manner. Due to the reaction of the
polyamine, for example urotropine, with the proton of the acid (for
example citric acid), an aldehyde and the corresponding ammonium
salt are formed. Here, an equilibrium is provided that is
influenced by the dissociation constant of the acid and the
hydrolysis constant of the polyamine and here adjusts the pH to a
certain level-at approximately pH 4 to pH 8. In the case of
urotropine, 1 mol of urotropine can consume 6 mol of protons,
whereby even small quantities of urotropine or polyamines buffer
large quantities of acid, and therefore the pH of the solution is
held at a constant value.
[0043] Due to this stability, pH fluctuations can be practically
excluded. For example a pH change by formation of formic acid is
thus also avoided, and therefore it is also impossible to find any
"formalin pigments" with use of the solution according to the
invention. Additives known to a person skilled in the art for
stabilising solutions, which additives could interfere with a
stain, such as methanol and butanol, therefore fortunately also are
unnecessary.
[0044] A further advantage of the solution according to the
invention is the colour brilliance and colour intensity of the
samples fixed using the solution according to the invention. Thus,
practical examples 4-5 for the fixations performed by way of
example demonstrate a significant improvement of the morphological
preservation and cuttability of the fixed samples. Furthermore, in
the case of the performed stains, specifically haematoxylin &
eosin, Masson-Goldner Trichrome, MSB-Lendrum and Azan according to
Geidies, described in examples 4 and 5, it is shown that a
significant improvement of the stainability in general and in
particular of the colour saturation and of the colour brilliance is
attained by use of the solution according to the invention for
fixation. In addition, numerous tissues that are difficult to
process, such as brain, skin and testes, can be much better cut and
stained with the new fixation.
[0045] It is assumed, without introducing a limitation as a result
of this assumption, that the improvement of the stainability, even
with a small addition of the multi-functional aldehydes, such as
citral, 3,7-dimethylocta-2,6-dienal, geranial,
(E)-3,7-dimethylocta-2,6-dienal, propanal, butanal, valeric
aldehyde, pentanal, hexanal, heptanal, (2 E)-3-phenylprop-2-enal,
benzaldehyde, phenylmethanal, vanillin aldehyde,
4-hydroxy-3-methoxyphenylmethanal, heptanal, octanal, nonanal,
decanal, and mixtures thereof can be attributed to the fact that
they are added together in the balanced reaction.
[0046] In the declared solution, such a balanced reaction takes
place, in which the polyamine with the protons released by the acid
transitions for example into an aldehyde. The added, additional
multi-functional aldehydes are summed on the aldehyde side and can
then also be incorporated in the sample. The multi-functional
groups of these aldehydes serve here as additional docking points
for dyes, which can be bound to these points via polar bonds,
hydrogen bridges or even covalent bonding, and thus improve the
colour reactions of the histological and/or immunohistochemical
stains used.
[0047] It has fortunately been found that further problems, which
are known with other formalin-free fixations, are also evaded or
avoided by the use of the declared solution.
[0048] Examples include the HOPE fixation (Hepes-Glutamic acid
buffer mediated Organic solvent Protection Effect), which is
particularly suitable for molecular biological questions, since
nucleic acids and antigen structures are preserved particularly
well by means of this fixation agent. However, this fixation can be
performed only using complex apparatus and by means of a complex
processing procedure, and therefore is only suitable with
difficulty for daily routine.
[0049] Another formalin-free fixation agent is offered by the
company Anatech as "prefer fixative". This contains glyoxal,
ethanol and buffer; here, however, only few experience values have
so far been presented with regard to the histological application.
The presence of alcohol in the solution already rules it out,
however, for numerous stains.
[0050] A further product originates from the company Sigma and is
distributed under the name Accustain. Here too, ethanol is the main
component of the fixation agent.
[0051] Currently, a comprehensive testing and economical evaluation
of the results of histological stains is not available for any of
the alternative fixation agents. The demands on a formalin-free
fixation agent are primarily a comparability of the result with
previous results of formalin-fixed samples, and identical or
improved handling as a matter of routine, identical or similar
fixation periods, and identical or improved staining
properties.
[0052] The solution according to the invention meets these demands
and for the first time allows the fixation and staining of tissue
samples fixed in a formalin-free manner whilst maintaining and even
considerably improving the colour saturation, colour brilliance and
colour intensity in the methods listed hereinafter: [0053] a)
Haematoxylin & Eosin staining, with use of various haematoxylin
solutions (according to Mayer, Gill, Harris, Weigert, Verhoff,
Hansen, etc.) and Eosin solutions (in aqueous, alcohol or methanol
form with various concentrations and additives of acetic acid or
other pH-lowering additives). [0054] b) Trichrome staining, for
example according to Masson, Masson-Goldner, Azan, Crossmon,
Mallory, Cason, and other staining protocols which attain a
single-, two-, three- or multi-stage stain via nuclear stains,
plasma stains and fibre stains. [0055] c) Tri-, tetra-, penta- and
poly-chromatic overview and special stains, such as Movat
pentachrome, Mollier quadruple staining, van Gieson, Hansen,
Weigert and other stains for the physiological or chemical staining
of special tissue structures. [0056] d) Special stains or special
detections of fibres, tissue components, cell nuclei, plasma
components and chemical properties, such as elastica stains,
aldehyde detections, iron detections, other metal detections,
amyloid detections, fat staining, representation of
mucopolysaccharides, silver stains and gold toning, selective
nucleus stains, cytological and haematological stains, calcium
detections, bone and cartilage stains, nerve stains, etc.
[0057] For all mentioned staining methods, it should be ensured
that, subsequent to the fixation, the tissue pieces placed in the
fixation agent or tissue pieces, organs or entire organisms
saturated with the fixation agent are subjected to a conventional
further processing that is routine in the laboratory, that is to
say they are dewatered and then infiltrated with paraffin or other
embedding media (celloidin, acrylates) and processed to form sample
blocks, which can ultimately be cut using a microtome.
[0058] Here, the invention improves the plastic properties of the
fixed material, such that, when processing thin sections, cutting
artefacts are absent or only occur to a small extent, because on
the one hand there is no tissue hardening caused by "overfixing"
and on the other hand the tissues do not shrink to such an extent
as is usual in the case of formalin fixations.
[0059] Here, it has been found that the tissue pieces treated by
means of the invention can be processed comparatively to, and
sometimes even more easily than formalin-fixed tissue samples. A
further advantage of the solution according to the invention is
therefore the fact that tissue samples that have been fixed using
this solution demonstrate excellent cutting properties, and, with
such tissues that are difficult to cut when they have been fixed
using solutions containing formaldehyde (for example brain tissue),
there are considerable improvements to the cutting properties.
[0060] Fewer artefacts are therefore introduced into the
histological section, which enables a significant improvement of
the microscopic examination of the sample and therefore
considerably facilitates the diagnosis of histological anomalies
and abnormalities.
[0061] As a result, the stains can be differentiated not only
identically, but much more firmly and much better compared with
stains on formalin-fixed samples, since colour differences in the
histological section are sharper and more vibrant than is the case
with formalin-fixed samples, and the colours themselves are
brighter and more selective for differences in the chemism of the
tissue.
[0062] The tissue preservation is additionally advantageous.
Shrinkage hardly occurs, and when it does it is to a much smaller
extent than with formalin fixations. Cell nuclei retain their round
shape and can be stained very well using conventional nucleus dyes.
Even in the finest of structures, an excellent preservation can be
observed, which even equals the preservation of fixations using
fixation agents containing sublimate and picric acid.
[0063] The simple handing is also advantageous. The established
laboratory routines do not have to be changed, since fixing times
and application can remain identical. Tissue samples are introduced
into the sample containers with the invention and remain there
until further processed. The fixing times correspond to those of
formalin. Longer fixations are not problematic and may even be
advantageous. However, an unwanted overfixing of the tissue during
treatment with the solution according to the invention should not
be observed in any case. Further, the declared solution is harmless
in terms of hazardous materials and dangerous substances and can
also be handled as harmless chemical waste with regard to
disposal.
[0064] The invention furthermore also relates to the use of the
solution as a preservative for macroscopic preparations, that is to
say including entire animal bodies or corpses, as are used in the
field of anatomical science. In addition, it can also be used for
the fixing and long-term preservation of biological sample material
in natural history museums, zoological or botanical collections,
research collections and teaching collections.
[0065] The invention also relates to the use of the fixation agent
for immunohistochemical stains with paraffin-conventional
antibodies and also for antibodies that are suitable for frozen
sections or for plastic preparations.
DESCRIPTION OF THE FIGURES
[0066] FIG. 1: shows a comparison of staining results of an AZAN
stain on a tissue sample, specifically rat cerebellum, wherein the
sample used for FIG. 1A was fixed using formalin 4% in accordance
with standard methods and the sample used for FIG. 1B was fixed in
an identical method, but using the solution according to the
invention. It can be clearly seen that FIG. 1B has fewer cracks,
demonstrates an improved preservation of the preparation and of the
neurons, and also adopts a more vibrant staining.
[0067] FIG. 2: shows a comparison of staining results of a
HAEMOTOXYLIN & EOSIN (H&E) stain on a tissue sample,
specifically rat testes, wherein the sample used for FIG. 2A was
fixed using formalin 4% in accordance with standard methods and the
sample used for FIG. 2B was fixed in an identical method, but using
the solution according to the invention. It can be clearly seen
that FIG. 2B has clear delimited structures, fine structures are
better preserved, and the sample adopts a more vibrant
staining.
[0068] FIG. 3: shows a comparison of staining results of an MSB
LENDRUM stain on a tissue sample, specifically rat testes, wherein
the sample used for FIG. 3A was fixed using formalin 4% in
accordance with standard methods and the sample used for FIG. 3B
was fixed in an identical method, but using the solution according
to the invention. It can be clearly seen that in FIG. 3B the
differentiation of the fine structures is better, and the sample
adopts a more vibrant staining.
[0069] FIG. 4: shows a comparison of staining results of a MOVAT
stain on a tissue sample, specifically rat lung, wherein the sample
used for FIG. 4A was fixed using 4% formalin in accordance with
standard methods and the sample used for FIG. 4B was fixed in an
identical method, but using the solution according to the
invention. It can be clearly seen that FIG. 4B has clearer
delimited structures, fine structures (bronchi and alveoli) are
better preserved, and the sample adopts a more vibrant
staining.
[0070] FIG. 5: shows a comparison of staining results of a MASSON
TRICHROME stain on a tissue sample, specifically rat tongue,
wherein the sample used for FIG. 5A was fixed using formalin 4% in
accordance with standard methods and the sample used for FIG. 5B
was fixed in an identical method, but using the solution according
to the invention. It can be clearly seen that FIG. 5B has a sharper
differentiation of the fine structures, the stain leads to cell
nuclei stained more vibrantly, and the sample as a whole adopts a
more vibrant staining.
EXAMPLES
Example 1
Composition and Component of a Fixation Agent
TABLE-US-00001 [0071] POS. PRODUCT % G/L Use Range 1 Urotropine 4
40 up to 20% 2 Citric acid 5.43 54.29 up to 35% 3 TWEEN 80 1.43
.times. 10E-2 0.143 up to 5% 4 Monopropylene glycol 2.86 28.57 up
to 20% 5 (2 E)-3-phenylprop-2- 4.29 .times. 10E-2 0.429 up to 5%
enal 6 SPAN 80 0.71 .times. 10E-2 0.07 up to 5% 7 H.sub.20 or
suitable solvent. 87.65 876.5 filling to 100% 8 TOTAL 1000
Example 1.1
Calculation of the Molar Ratios of the Fixation Agent to Tissue
Fixation
[0072] With a composition according to Example 1 with the
components urotropine and citric acid, the molar ratio is
calculated as follows:
[0073] Urotropine releases up to 6 mol formaldehyde from 1 mol
urotropine. Citric acid releases up to 3 mol protons from 1 mol
citric acid. 1 mol citric acid (anhydrous)=192.124
g/mol=>192.124 g; furthermore, 1 mol urotropine=140.19
g/mol=>140.19 g.
[0074] A solution that contains 4% urotropine and 5.5% citric acid
(as proposed in Example 1) thus achieves a molar concentration of
urotropine of 0.28 M and can form at most up to 1.71 M
formaldehyde. Further, the molar concentration of citric acid is
0.28 M and can form at most up to 0.85 M protons.
[0075] This means that, in the solution from Example 1, 0.85 mol
formaldehyde can be formed from HMTA and an excess of HMTA also
remains in the solution. The molar ratio between total releasable
aldehyde and total protons releasable from the acid is in this case
approximately 2:1, wherein it must be taken into consideration that
urotropine is present in excess.
[0076] In the case of a balanced reaction, in which 0.85 mol
formaldehyde can be formed, the solution functions excellently as a
fixation solution for histology (see Example 5 and FIGS. 1 to
5).
1.2. Comparative Calculation: On the Example of Another
Conventional Solution:
[0077] A known solution containing 1% hexamethylenetetramine (HMTA)
and 1% citric acid, in accordance with the corresponding
calculation as under Example 1.1., has a molar concentration of
HMTA of 0.07 M and can form at most up to 0.42 M formaldehyde.
Here, the molar concentration of citric acid is 0.052 M and can
form at most up to 0.156 M protons.
[0078] This means that, with this known solution, at most 0.156 mol
formaldehyde can be formed from HMTA and an excess of HMTA remains
in the solution. The molar ratio between total releasable aldehyde
and total protons releasable from the acid is in this case
approximately 2.6:1.
[0079] If a maximum of 0.156 mol formaldehyde is formed, the
solution does NOT function as a fixation solution for histology,
since all known side-effects occur, as also described in the text
above, such as shrinkage and morphological changes up to the onset
of degeneration of the tissue. The aforementioned values were
confirmed by way of experiment for HMTA and citric acid.
Example 2
Composition and Components of a Further Preferred Embodiment
TABLE-US-00002 [0080] PRODUCT % G/L Use Range Hexamethylol melamine
6 60 0-20% Boric acid 3 30 0-35% Sodium hydroxide 0.4 4 0-5%
Dipropylene glycol 2.86 28.57 0-20% Phenylmethanal 0.01 0.1 0-5%
Polysorbate 20 0.01 0.1 0-5% H.sub.20 or suitable solvent 87.65
876.5 10-99% TOTAL 1000
[0081] Hexamethylol melamine, referred to hereinafter as HMM,
enters into the following balanced reaction:
##STR00001##
[0082] HMM
[0083] This reaction best takes place at pH 6-8, which is why a
weak acid, specifically boric acid, is used in accordance with the
present embodiment. The pH is adapted with sodium hydroxide. It may
be that ->6 mol formaldehyde are released per mol HMM. This
gives:
TABLE-US-00003 1 mol HMM 270 g 1 mol formaldehyde 30 g 1 mol HMM
contains 6 mol aldehyde or 180 g In order to obtain a solution with
total releasable formaldehyde content of 4%/l, 40 g releasable
formaldehyde or 60 g HMM are consequently required.
[0084] Three protons are cleaved from the boric acid. Boric acid
can be represented by the formula H.sub.3BO.sub.3 and has a molar
mass of 61.83 g. In order to obtain 6 mol of protons, 27.5 g are
therefore required, that is to say 30 g of boric acid calculated
with slight excess, based on 1000 ml solution.
[0085] In the present composition, a ratio of total releasable
aldehyde/total releasable protons of approximately 1:1 is
produced.
Example 3
Further Alternative Embodiments
[0086] In accordance with the calculations presented above in
Example 1.1 and also Example 2, further alternative compositions
can be prepared. The components that can be used alternatively are
specified in the table below.
[0087] Any combination of the substances specified under Position 1
with the substances from Position 2 is possible. Where appropriate,
the substances from Position 5 are combined, for example in order
to thus adjust the proportion of total releasable aldehyde. In
accordance with the invention, a solution formed by these
combinations must comprise at least 0.5 mol/l of total releasable
aldehyde. The substance alternatives of Positions 3 and 4 and also
6 and 7 are optional and can be combined as necessary.
TABLE-US-00004 POSITION based on the table in Example 1 ALTERNATIVE
COMPONENTS 1 urotropine, triazines, dimethylol, dihydroxyethylene
urea, tetramethylol acetylene diurea, mono-, di-, tri-, tetra-,
penta- or hexamethylol melamine, dimethyl propylene urea,
acetoguanamine or 5,5-dimethyl- hydantoin, or mixtures thereof 2
oxalic acid, tartaric acid, succinic acid, hydrochloric acid,
acetic acid, propionic acid, formic acid, mono-, di- or
trichloroacetic acid or chloroacetic acid, uric acid, citric acid,
boric acid, phosphoric acid, or mixtures thereof 3 TWEEN 80,
polysorbate 20, 40, 60 or 80, alkalines, lauryl sulfates; dodecyl
sulfates, dodecyl sulfonates, dodecylbenzene sulfonates;
ethoxylated fatty acids; ionic and non-ionic surfactants with high
HLB (hydrophilic-lipophilic balance) value, (in chemistry describes
the hydrophilic and lipophilic proportion of primarily non-ionic
surfactants and was proposed in 1954 by W. C. Griffin. Surfactants
with a high HLB value cause a good wetting of hydrophilic
surfaces), or mixtures thereof 4 mono-, di- or further
polypropylene glycols; glycerol, mono-, di-, or further
polyethylene glycols; sorbitol, pentaerythritol, low-molecular PEG,
high-molecular PEG, and mixtures thereof 5 citral,
3,7-dimethylocta-2,6-dienal, geranial,
(E)-3,7-dimethylocta-2,6-dienal, propanal, butanal, valeric
aldehyde, pentanal, hexanal, heptanal, octanal, nonanal, decanal,
(2 E)-3- phenylprop-2-enal, benzaldehyde, phenylmethanal, vanillin
aldehyde, 4-hydroxy-3-methoxyphenyl- methanal, and mixtures thereof
6 SPAWN 80, polysorbates, lauryl sulphates, ethoxylated fatty
acids, ionic and non-ionic surfactants with low HLB, or mixtures
thereof 7 H.sub.2O; alcohols; acetones; dimethyl sulfoxide; alkyl
carbonates; polar organic solvents, and mixtures thereof
Example 4
Method for the Preparation of Histological Samples for Microscopic
Examination
[0088] In accordance with the inventive method, the tissue sample
should be introduced as fresh as possible into the fixation
solution, for example the solution according to Example 1.
Generally, it is usual for the quantity of fixation solution to
correspond to twenty times the sample volume and for the fixation
period to be dependent on the sample size. Depending on size, the
samples remain in the fixation solution from 12 to 36 hours, and
larger samples may also remain in the fixation solution for 72
hours or longer. A diffusion distance or penetration rate of 1 mm
in 2-4 hours is assumed.
[0089] In order to then embed the sample in paraffin or other
embedding agents, a complete dewatering of the sample is necessary.
The sample is first removed from the fixation solution, washed in
flowing water and immersed in a series of alcohols in rising
concentration. The steps between the concentration stages are here
30-50-60-70-80-90-96-100, for example. Ethanol or isopropanol are
usually used as alcohols. Following the dewatering, the sample is
immersed in a suitable organic solvent, which is miscible both with
alcohol and with the embedding medium, then the infiltration with
the actual embedding medium (paraffin) follows in a number of
stages, in each case for a number of hours (depending on the sample
size). The sample is cast into a suitable mould with a small
quantity of paraffin. Once the block has solidified, the block is
removed from the mould and then can be cut. Using a suitable
microtome, sections 4-6 .mu.m thick are produced and are arranged
on glass slides.
[0090] The glass slides with the paraffin sections can be stored
for a relatively long period of time. However, further processing,
that is to say staining, generally occurs directly. Here, the
paraffin first has to removed again and the tissue section itself
watered. The procedure previously described is performed in the
reverse order, that is to say the paraffin is dissolved out using
xylene, followed by alcohol 96%, 90%, 80%, 70% or a similar
sequence up to water. The staining then starts, wherein, in the
case of dyes that are present in alcohol solution, the steps up to
complete watering can be omitted and the glass slides can be
immersed directly into the alcohol staining solution.
Example 5
Assessment of the Morphological Preservation, Cuttability and
Stainability of Paraffin Preparations
[0091] With the aid of conventional histological stains, it was
possible to carry out a diagnostic evaluation of section samples.
The sections were produced from tissues which had previously been
fixed either in the solution from Example 1 or in 4% formalin. The
qualitative evaluation was performed on four stains selected by way
of example: (1) haematoxylin & eosin (HE), (2) Azan according
to Geidies, (3) Masson Goldner Trichrome and (4) MSB Lendrum. The
histological diagnostic evaluation detects the parameters
morphological preservation and stainability of the sections. The
parameter cuttability was assessed during the cutting process
performed on various tissues.
Stains Used by Way of Example
[0092] HAEMOTOXYLIN & EOSIN: the conventional H & E stain
uses the dyes haematoxylin in the composition according to Mayer
and eosin. Both dyes are offered in aqueous solution (eosin
possibly also as alcohol solution) with a defined pH value and/or a
defined acid addition and provide a characteristic colour image.
Cell nuclei are generally stained blue-purple here. In this case,
the dye haematoxylin, which is offered as Alaun complex, reacts
with the chromatin of the cell nuclei. In order to obtain a stable
water- and alcohol-insoluble coating, this compound is then
immersed in tap water or in a salt solution mixed with monovalent
metal ions. The eosin solution is set in aqueous form to a pH value
of approximately 4.0, which leads to a characteristically
illuminating orange colour image. Eosin binds in different
intensity to various tissue and cell structures and thus allows a
good differentiation of the histological section.
[0093] MASSON GOLDNER TRICHROME: the Masson Goldner Trichrome and
the Crossman stain are also conventional histological stains which
function as Trichrome staining with three (or four) dyes. Here too,
the nuclei are stained with haematoxylin, but not with an Alaun
complex, but via an iron complex. This does not lead to a cell
nucleus stained blue-purple, but to a brown-black cell nucleus,
which can be differentiated very clearly, even with respect to
tissue components stained blue. A mixture of acid fuchsin, ponceau
and azophloxine is used as a second dye and binds differently to
different tissue regions and makes it possible to identify cell
plasma and other intracellular structures. This dye complex reacts
directly with the respective tissue structures. A further dye,
Orange G, is what is known as a mordant dye, which forms a water-
and alcohol-insoluble coating with certain tissue structures only
in the presence of a bivalent metal ion, for example as is provided
by phosphotungstic acid or phosphomolybdic acid. In particular,
erythrocytes and muscles can be presented using this dye and can be
significantly distinguished from other tissues. Keratinous material
is also stained considerably with Orange G. Alternatively, aniline
blue or light green is used as a fourth dye. These are conventional
fibre dyes, which bind particularly with collagens and
intercellular structures. Cartilage and bone material also bind to
these dyes, depending on chemism. Different blue or green
intensities ultimately allow diagnostic conclusions concerning the
fibre composition, wherein aniline blue generally provides the
differentiated image.
[0094] MSB LENDRUM: the MSB Lendrum stain is a stain related to the
Masson Goldner Trichrome, in which, however, the dyes Martius
yellow and crystal ponceau are offered instead of acid
fuchsin/azophloxine/ponceau and instead of Orange G. This results
in an image that is comparable, but differentiated further still,
wherein the significance of the MSB staining is of particular
value, especially for vascular structures, because muscles, fibre
arrangement and vessel inner walls are better differentiated here
than with the previously mentioned Trichrome stains.
[0095] AZAN ACCORDING TO GEIDIES: the AZAN stain also belongs to
the series of conventional Trichrome staining. The original variant
according to Heidenhain works in the pre-treatment of the sections
with aniline alcohol, azocarmine for nucleus staining,
phosphotungstic acid for staining and a dye mixture of aniline blue
and Orange G and lasts for a number of hours. By contrast, the
variant modified according to Geidies dispenses with aniline
alcohol, and instead uses the azocarmine nuclear fast red and
drastically reduces the staining times. The result of the stain is
so similar that the conventional AZAN stain can be largely
replaced. The results are substantially comparable to those of the
Masson Goldner and MSB Lendrum. Cell nuclei are stained deeply red
by the nuclear fast red provided in aluminium sulfate solution and
contrast well with the continuous blue background. Aniline blue and
Orange G produce the counter or background stain (blue) and a well
differentiated staining of the erythrocytes (orange) and of the
muscle plasma (also deeply orange). In addition, creatine and
callous epithelia are also stained significantly orange. The basic
substance and fibres obtain different blue shades as a result of
the aniline blue. The result of the aniline blue stain is
influenced on the whole very strongly by the stain duration.
5.1 Morphological Preservation
[0096] In order to assess the morphological preservation at light
microscopy level, the following parameters can be used: (A)
fragmentation of the tissue (crack formations) and (B) the
presentation of the cell nuclei (nuclei) and (C) of the cell bodies
(stomata).
[0097] The fragmentation of the tissue by the formation of
individual cracks or propagated crack networks results from
deficient fixing and manifests itself as the tissue section is
drawn on the hot water bath or the hot plate and also during the
staining process. During the course of the heat-induced drawing of
the paraffin and therefore also of the tissue section penetrated
and covered by paraffin, a physical loading of the paraffin/tissue
association is produced, which may result, in the case of deficient
fixing, in the breaking of the tissue. The tensile and/or shear
forces occurring during deparaffinisation and staining of the
tissue section are applied particularly to these tissue points,
which are already strained during the drawing process, expand these
tissue points or break the tissue entirely. The tendency for crack
formation is proportional to the adhesive property of the slide
used.
[0098] The score is awarded between (no fragmentation of the
tissue, 1) and (cell nuclei and cell bodies in their natural form,
1) and (tissues destroyed by crack formation and detached, 4) and
(cell nuclei angular and severely shrunken, cell body massively
shrunken with strong "ring formation", 4).
General Criteria
TABLE-US-00005 [0099] Fragmentation Score Cell nuclei Score Cell
body Score No 1 Natural 1 Natural 1 fragmentation Slight 2 Slightly
2 Minimal 2 fragmentation angular shrinkage Moderate 3 Moderately 3
Moderate 3 fragmentation angular shrinkage Extreme 4 Cell nucleus 4
Massive 4 fragmentation: angular and shrinkage tissue is severely
and/or ring destroyed over shrunken formation a large area
5.2 Cuttability
[0100] A further criterion in the histological processing of tissue
samples is what is known as the cuttability of the tissue. The
different properties of fixation agents result, usually in
accordance with the incubation time, in a different cuttability or
load-bearing capacity of the tissue. A tissue, for example, can be
brittle and fragile (no cuttability, 4) or supple and resistant
(high cuttability, 1) as a result of the fixation. Different tissue
properties of different tissue types have to be taken into
consideration in the assessment.
General Criteria
TABLE-US-00006 [0101] Cuttability Score High cuttability 1
Above-average cuttability 2 Below-average cuttability 3 No
cuttability 4
5.3 Stainability
[0102] Histological stains of tissue sections often allow various
distinctions between individual cells, but also tissue types,
depending on the dye combinations used. This histological
differential diagnosis applied in human and veterinary diagnostics,
but also in medical, zoological and botanical research, is based on
the differentiation of nucleus types, but in particular also on the
differentiation of dye precipitation on cells and/or tissues. A
maximum dye saturation, uniform colour precipitation, good
contrasting (between cell nucleus and cell body) and also a high
colour brilliance are therefore not only desirable, but also
absolutely necessary requirements of a stain.
[0103] Chemical reactions between the fixation agent and the tissue
influence not only the cuttability and the load-bearing capacity of
the tissue, but also the stainability thereof. Different fixation
agents can improve, but also reduce the stainability of a tissue
section depending on the incubation time. In order to determine the
stainability of tissues, the following criteria are used: 1. colour
saturation, 2. uniform colour precipitation, 3. contrast between
cell nucleus and cell body and 4. colour brilliance. The individual
stains have to be assessed separately from one another, since
stains or components thereof respond differently to the influence
of fixation agents.
General Criteria
TABLE-US-00007 [0104] 1. Colour saturation Score Very good colour
saturation 1 Good colour saturation 2 Moderate colour saturation 3
Inadequate colour saturation 4
TABLE-US-00008 2. Colour precipitation Score Uniform colour
precipitation without oversaturation 1 Colour precipitation with
low fluctuations 2 Partly irregular colour precipitation 3
Completely irregular colour precipitation 4
TABLE-US-00009 3. Contrast (cell nucleus/cell body) Score Very good
contrast (generally good differentiability 1 of nucleus components
and/or nucleus membranes) Good contrast (largely good
differentiability of nucleus 2 components and/or nucleus membranes)
Moderate contrast (little differentiability of nucleus 3 components
and/or nucleus membranes) Poor contrast (no differentiability of
nucleus 4 components and/or nucleus membranes)
TABLE-US-00010 4. Colour brilliance Score Very high colour
brilliance 1 High colour brilliance 2 Low colour brilliance 3 Very
low colour brilliance 4
5.4 Evaluation/Result
[0105] The described criteria catalogue forms the basis for the
presentation below of the results in table form. Section samples
were produced following a 24/hr fixation period in the solution
according to Example 1 or 4% formalin from the following tissues:
brain, heart, lung, kidney, skin and testes. These were then
stained simultaneously with HE, Azan according to Geidies, Masson
Goldner Trichrome and MSB Lendrum.
TABLE-US-00011 1. Morphological preservation: Solution according
Criterion to Example 1 Formalin Fragmentation 1 3 Cell nuclei 2 2
Cell bodies 2 2
TABLE-US-00012 2. Cuttability Solution according Tissue type to
Example 1 Formalin Brain 1 2 Heart 2 2 Lung 2 3 Kidney 2 3 Skin 1 2
Testes 1 2
TABLE-US-00013 3.a. Stainability (H & E): Solution according
Criterion to Example 1 Formalin 1. Colour saturation 1 2 2. Colour
precipitation 2 3 3. Contrast 1 2 4. Colour brilliance 1 2
TABLE-US-00014 3.b. Stainability (Azan according to Geidies)
Solution according Criteria to Example 1 Formalin 1. Colour
saturation 1 2 2. Colour precipitation 2 2 3. Contrast 2 2 4.
Colour brilliance 1 3
TABLE-US-00015 3.c. Stainability (Masson Goldner Trichrome):
Solution according Criteria to Example 1 Formalin 1. Colour
saturation 1 2 2. Colour precipitation 2 3 3. Contrast 1 2 4.
Colour brilliance 1 3
TABLE-US-00016 3.d. Stainability (MSB Lendrum): Solution according
Criteria to Example 1 Formalin 1. Colour saturation 1 3 2. Colour
precipitation 2 3 3. Contrast 2 4 4. Colour brilliance 2 4
* * * * *